Thrombin-induced delayed injury involves multiple and distinct signaling pathways in the cerebral cortex and the striatum in organotypic slice cultures

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Abstract

Thrombin, a serine protease essential for blood coagulation, also plays an important role in cellular injury associated with intracerebral hemorrhage. Here, we show that, in organotypic cortico-striatal slice cultures, thrombin evoked delayed neuronal injury in the cerebral cortex and shrinkage of the striatum. These effects were prevented by cycloheximide and actinomycin D but not by a caspase-3 inhibitor. Thrombin-induced shrinkage of the striatum was abolished by a thrombin inhibitor argatroban or prior heat inactivation of thrombin, and significantly attenuated by a protease-activated receptor-1 antagonist FR171113. However, thrombin-induced cortical injury was not prevented either by heat inactivation or by FR171113, and was only partially inhibited by argatroban. In addition, inhibition of extracelluar signal-regulated kinase (ERK), Src tyrosine kinase and protein kinase C prevented both neuronal injury in the cortex and shrinkage of the striatum, whereas inhibition of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase prevented shrinkage of the striatum only. Thrombin treatment promptly induced phosphorylation of ERK, which was not prevented by inhibition of Src and protein kinase C. Thus, thrombin induces cellular injury in the cerebral cortex and the striatum, by recruiting multiple and distinct signaling pathways in protease activity-independent as well as dependent manner.

Introduction

Thrombin, a serine protease generated from prothrombin at sites of vascular injury, plays an important role in blood coagulation and wound healing. Prothrombin mRNA is also expressed in various brain regions (Dihanich et al., 1991), which indicates that thrombin may play a role as a signaling molecule in the central nervous system. Indeed, members of protease-activated receptor (PAR) family that mediate biological actions of thrombin are distributed widely throughout the central nervous system. Thrombin cleaves amino terminal exodomain of PAR-1, -3 and -4, and the newly generated amino terminus binds to the extracellular domain of the receptor as a tethered ligand (Noorbakhsh et al., 2003). PARs are G protein-coupled receptors with seven transmembrane segments. Activation of these receptors leads to various kinds of intracellular signal transduction, including increase of intracellular calcium (Smith-Swintosky et al., 1995), decrease of cyclic AMP (Yang et al., 1997) and activation of protein kinase C (PKC) (Wang et al., 2002). In addition, several lines of evidence suggest that mitogen-activated protein kinases (MAPKs) are involved in thrombin-induced cellular responses (Marinissen et al., 2003, Suo et al., 2003, Wang et al., 2002).

At low concentrations, thrombin may provide cytoprotective effects on neurons and astrocytes against oxidative stress and ischemic injury (Donovan and Cunningham, 1998, Striggow et al., 2000, Vaughan et al., 1995). By contrast, uncontrolled thrombin activity after intracerebral hemorrhage results in gliosis and neural cell death. For example, experimental evidence in vitro has shown that thrombin causes neurite retraction in differentiated neural cell lines (Jalink and Moolenaar, 1992), and cell death in dissociated neuron cultures and cultured hippocampal slices (Donovan et al., 1997, Striggow et al., 2000, Suo et al., 2003). Intrastriatal injection of thrombin causes neuronal degeneration in vivo (Xue and Del Bigio, 2001), and neuronal injury and neurological dysfunction associated with intracerebral hemorrhage are ameliorated by argatroban, a thrombin inhibitor (Kitaoka et al., 2002). Moreover, thrombin may also play a pivotal role in other pathological conditions including ischemic stroke, traumatic injury and Alzheimer's disease (Christov et al., 2004, Xi et al., 2003).

Despite these lines of evidence, little information is available concerning signaling mechanisms mediating cytotoxic actions of thrombin. Thrombin-induced neuronal death exhibits features of apoptosis (Donovan et al., 1997, Smirnova et al., 1998, Turgeon et al., 1998), and caspase inhibitors attenuate thrombin-induced cell death (Choi et al., 2003a, Turgeon et al., 1998). Activation of extracellular signal-regulated kinase (ERK), a kind of MAPKs, appears to be involved in thrombin-induced cell death in primary hippocampal neuron cultures (Suo et al., 2003). On the other hand, in the midbrain substantia nigra, thrombin induces degeneration of dopaminergic neurons indirectly via activation of microglia, which may involve several members of MAPK family such as ERK, p38 MAPK and c-Jun N-terminal kinase (JNK) (Choi et al., 2003a, Choi et al., 2003b, Lee et al., 2005). Contribution of multiple signaling mechanisms may differ among different experimental conditions and cellular contexts.

Intracerebral hemorrhage occurs preferentially in several brain regions including the cerebral cortex and the striatum, but the detailed mechanisms of thrombin-induced injury in these brain regions remain to be determined. In this study, we investigated cytotoxic consequences of thrombin application to organotypic cortico-striatal slice cultures.

Section snippets

Drugs and chemicals

Drugs and chemicals were obtained from Nacalai Tesque (Kyoto, Japan), unless otherwise indicated. Thrombin from bovine plasma (catalog No. T4648) was obtained from Sigma (St. Louis, MO, USA). Three different lots (022K7604, 061K7612 and 023K7602) of thrombin gave similar results. Bovine serum albumin (catalog No. A2153), MK-801, aminoguanidine, Nω-nitro-l-arginine methyl ester (l-NAME), N-acetylcysteine (NAC) and clodronate were also obtained from Sigma. Cell-permeable DEVD-CHO, PD98059, U0126,

Thrombin induces delayed cortical neuron death and striatal degeneration

Cortico-striatal slices were maintained for 9–11 days in culture. As previously described (Fujimoto et al., 2004), the striatal region gradually flattened during cultivation and typically left monolayer of cells, whereas the cortical region maintained much thicker appearance than the striatal region. These cultures were exposed to thrombin at concentrations of 10–300 U/ml for 72 h, and PI fluorescence was used as a measure of cell injury. We could also observe the entire morphology of

Discussion

Accumulating evidence indicates that thrombin exerts deleterious effects in the central nervous system under several pathological conditions including intracerebral hemorrhage (Gingrich and Traynelis, 2000, Xi et al., 2003). Because thrombin receptors are expressed not only in neurons but also in astrocytes and microglia (Grabham and Cunningham, 1995, Suo et al., 2002), thrombin extravasation should mobilize complicated processes that lead to cellular injury. In this study, we used organotypic

Acknowledgments

This study was supported by Grant-in-aid for Scientific Research from The Ministry of Education, Culture, Sports, Science and Technology, Japan and Japan Society for the Promotion of Science.

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